Abstract
Nitric oxide (NO) is a key signaling molecule in biological systems. New tools are required to therapeutically modulate NO levels with confined precision. This study explores the photoactivatable properties of an NO releasing compound (CPA), based on cupferron O-alkylated with an anthracene derivative. Upon light stimulation, CPA uncages two species: cupferron, which liberates NO, and an anthrylmethyl carbocation, which evolves into a fluorescent reporter. Proof-of-principle is demonstrated using one- and two-photon excitation (1PE and 2PE) in a cellular system (A431 cells). It was found that 1PE induces cell toxicity, while 2PE does not. Since 1PE using UV light is more likely to generate cellular photodamage, the cell toxicity observed using 1PE is most likely a combinatory effect of NO release and other UV-induced damage, which should be subject to further investigation. On the other hand, absence of phototoxicity using 2PE suggests that NO alone is not cytotoxic. This leads to the conclusion that the concept of 2PE photorelease of NO from CPA enable opportunities for biological studies of NO signaling with confined precision of NO release with minimal cytotoxicity.
Highlights
Nitric oxide (NO) is a key signaling molecule in biological systems
In particular we demonstrate that i) photoexcitation of 1 can be performed in a cellular system under biological conditions; ii) the formation of fluorescent co-product 2 (Fig. 1) can be traced in a biological system functioning as an optical reporter for NO release; and iii) that NO induced cell mortality appears to be dependent on the photoexcitation modality (i.e. 1PE versus 2PE)
We explore the potential of obtaining light stimulated NO release using a dual-function photocaged compound, which upon light activation becomes fluorescent simultaneously as NO is released
Summary
Nitric oxide (NO) is a key signaling molecule in biological systems. New tools are required to therapeutically modulate NO levels with confined precision. Absence of phototoxicity using 2PE suggests that NO alone is not cytotoxic This leads to the conclusion that the concept of 2PE photorelease of NO from CPA enable opportunities for biological studies of NO signaling with confined precision of NO release with minimal cytotoxicity. One possibility to address this task is to use a fluorescent reporter This strategy relies on the simultaneous photo-release of the desired caged bioactive species and a fluorescent component from the same non-fluorescent caged precursor[18,19,20]. An antrylmethyl carbocation is simultaneously formed as key intermediate in the photodecomposition The precursor for this photo-reactivity pathway was demonstrated to be a charge transfer state which is responsible for the strong fluorescence quenching of the anthracene moiety[22]. In particular we demonstrate that i) photoexcitation of 1 can be performed in a cellular system under biological conditions; ii) the formation of fluorescent co-product 2 (Fig. 1) can be traced in a biological system functioning as an optical reporter for NO release; and iii) that NO induced cell mortality appears to be dependent on the photoexcitation modality (i.e. 1PE versus 2PE)
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